It is estimated that the human body may contain over two million proteins, yet these two million proteins are encoded by approximately only 20,000 to 25,000 genes. The discrepancy between the amount of proteins and the genes encoding those proteins can be explained by genes (or germline DNA) that contain a multitude of different coding sequences (exons) that are interspersed with non-coding sequences (introns) and how the splicing process takes copies of this germline DNA apart and puts it back together again. During the process of transcription, the genetic information in a DNA molecule is transferred to a primary RNA transcript. A primary RNA transcript is a copy of the DNA molecule in that it also contains exons interspersed with introns. The primary RNA transcript is then processed via “cutting out” introns, and many of the exons as well, and re-joining the pieces to create a messenger RNA (mRNA) with a unique combination of exons. The mRNA is then translated into a protein.
The term used to describe the cutting and re-joining process is “splicing.” Based upon where the splices are made, many different mRNA sequences and thus different proteins can be made from the transcription of a single piece of germline DNA. FIG. 1 shows, for example, how alternative splicing of a single primary antibody RNA transcript can result in the production of antibodies that differ from one another.
Splicing takes place in a large complex, the spliceosome, which contains approximately 200 proteins and 5 small RNAs (U snRNAs) (See Wachtel and Manely, Mol. BioSyst, 5:311-316 (2009)). Accordingly, there are likely a large number of factors that control which exons are spliced out and which remain, and many of these factors continue to be poorly understood. There continues to be a general need in the art for the development of splicing constructs, cell lines and assays that can be used to elucidate these factors and their mechanisms of action. There is also a specific need to elucidate the mechanism of splicing specific constructs such as those associated with CD44.
CD44 is a glycoprotein and a cell-adhesion protein. CD44 has also been implicated as a lymphocyte homing receptor. The CD44 human gene contains 20 exons, 10 of which encode the membrane proximal extracellular domain (See Fox et al., Cancer Research 54:4539-4546 (1994)). These 10 exons have been termed v1-v10. At least 20 different isoforms of CD44 have been described that result from the differential, or alternative, splicing of these 10 exons. Id. at 4539.
Importantly, the CD44 variant domain 6 (CD44v6) isoform has been implicated in tumorigenesis, tumor cell invasion, and tumor metastasis (See Heider et al., Cancer Immunol. Immunother. 53:567-579, 567 (2004)). Intense and homogeneous expression of CD44v6 was reported for the majority of squamous cell carcinomas and a portion of adenocarcinomas of differing origin. Id. at 567. Nevertheless, the splicing mechanism of action that results in the production of CD44v6 remains poorly understood and there is a need in the art for alternative splicing constructs and cell lines that can be used to study the mechanisms of CD44v6 splicing specifically.
In addition to CD44, other splice variant proteins such as MDM2, BRCA1/BRCA2, PSA and numerous members of the FGF receptor family have been reported to be differentially expressed in tumor cells when compared to their normal counterparts (See Brinkman, B. M., Clin. Biochem. 37:584-594 (2004)). Since alternative splicing has been associated with the development and/or progression of several cancers, researchers have suggested the use of alternative splicing as a means of targeting the expression of therapeutic genes to tumor cells in vivo (See Hayes et al., Cancer Gene Therapy 9:133-141 (2002)).
Hayes et al. suggest the use of a splice activated gene expression vector using the CD44 isoform R1 that is selectively active in tumor cells and produces alkaline phosphatase. CD44R1 contains contiguous variant domain 8, variant domain 9, and variant domain 10. Once the alkaline phosphatase is produced by the tumor cell according the methods of Hayes et al., an inactive pro-drug, etoposide phosphatase, is administered to the tumor cell. The alkaline phosphatase is excreted from the tumor cell and acts on the inactive pro-drug to create the active drug, etoposide. The etoposide then kills the tumor cell that produced the alkaline phosphatase as well as the surrounding cells. The Hayes et al. article notes that a benefit of their system is the greatly enhanced efficacy of the treatment due to the diffuse nature of the pro-drug. Since the pro-drug is located outside the tumor cell and the drug activator is excreted from the cell, the toxic effect occurs outside the tumor cell and allows for bystander cell killing. Id. at 139. In other words, the Hayes et al. system allows for killing of tumor cells and other non-tumor cells that do not splice the CD44R1 alternative splicing construct nor express the alkaline phosphatase.
While bystander killing could be beneficial in certain instances, there is a need in the art for alternative splicing constructs that do not require secretion of a pro-drug activator and an additional administration of a pro-drug to achieve tumor cell killing. There is also a need for alternative splicing constructs that work in tumor cells that will not splice the CD44R1 construct described in Hayes et al. Splicing of the CD44R1 construct is limited to those cells that express CD44R1 naturally. Accordingly, there is a need in the art to provide other alternative splicing constructs and their uses for cancer treatment that 1) do not require multiple administrations of pro-drugs, 2) do not result in extensive bystander cell killing, and 3) function in cells other than those cells that express CD44R1 naturally. As mentioned above, there is also a need to develop assays, including high-throughput assays, that allow for the study of these other alternative splicing constructs.